JP6284500B2 - Method for producing high-titer and high-purity virus stock and method for using the same - Google Patents

Description

  The present invention relates to a method for producing a high-titer and high-purity virus stock and a method for using the same.

This application claims the benefit of US Provisional Patent Application No. 61 / 324,220, filed Apr. 14, 2010. The entire teachings of the above application are incorporated herein by reference.

  Biopharmaceuticals such as monoclonal antibodies, recombinant proteins, vaccines, blood products and livestock products contain a risk of transmission of infectious viruses. This is due to the fact that the raw material may be inherently contaminated with viruses. In addition, the biopharmaceutical manufacturing process is susceptible to viruses from external sources. As a result, biopharmaceutical manufacturers must incorporate sufficient virus clearance steps into their manufacturing process to ensure that the product does not contain contaminating viruses. For safety and regulatory reasons, it is inevitable to incorporate such a virus clearance process into the biopharmaceutical manufacturing process.

  Evaluation of the effectiveness of the virus clearance process in the manufacturing process is essential. The purpose of viral clearance assessment is to determine the ability of an industrial production process to inactivate and / or remove potential viral contaminants. Typically, the virus clearance process is evaluated and validated using a spike test in a scale-down model of a production scale process. However, the test results are greatly influenced by the purity and titer of the virus stock used in the virus clearance test. The purity and titer of the virus stock affects how well the scale-down model reflects the production scale process.

  For example, impurities in virus stock adversely affect the testing of small virus retention filters used in virus clearance processes. Impurities are incorporated as a result of the use of impure virus stocks in validation spike tests and do not reflect typical manufacturing processes in production scale processes. As a result of the presence of impurities, the test unit (eg, filter) is prematurely fouled, exhibits a change in fouling conditions, and adversely affects the fluid passage of the virus filter. As a result, the true throughput capability of the filter is underestimated, thus leading to improper sizing of production scale units. As a result, the production scale unit becomes excessive to compensate for the apparent low performance of the unit in the validation test. This increases production costs. In addition, this underestimates the actual virus purification capacity of the test unit in the production process. This often determines the maximum virus concentration that the virus stock titer can spike into the test material, and this maximum concentration when all viruses are effectively cleared by the test unit is the virus clearance of the test unit. This is to determine the limit of the declared value.

  Current production methods for virus stocks (eg, those used in spike tests) lack the ability to produce virus stocks with low impurity content. Furthermore, since the amount of virus spikes that can be added to the test system is limited by regulatory guidelines, a virus stock with the highest possible titer is desirable. Current methods lack the ability to produce high titer and high purity virus stocks. Therefore, there is a need for a method of preparing a virus stock that is highly pure and has a high titer.

  Described herein are methods for producing high purity virus stocks, and viruses and virus stocks produced by such methods. The virus may be any suitable virus as required, such as a mammalian virus or a bacteriophage. Such virus stocks are suitable for use as virus spikes in, for example, evaluation and / or validation tests.

  Accordingly, one embodiment of the present invention is a method for preparing a high purity virus. The method includes sequential steps of obtaining a virus sample prepared under low protein conditions, concentrating the virus sample in a suitable buffer, and precipitating the virus in the virus sample. The virus thus precipitated is a high purity virus. In one embodiment, the high purity virus is a high purity virus stock solution.

  In another embodiment of the invention, the method for preparing a high purity virus comprises chromatographic polishing. In one embodiment, a method for preparing a high purity virus comprises obtaining a virus sample prepared under low protein conditions, concentrating the virus sample in a suitable buffer, and precipitating the virus in the virus sample. A sequential step of steps and chromatographic polishing of the precipitated virus, thereby producing a high purity virus. In one embodiment, the high purity virus is a high purity virus stock solution.

In one embodiment, the high purity virus or high purity virus stock solution has a protein concentration of less than 50 ug / ml. In another embodiment, the high purity virus or high purity virus stock solution has a protein concentration of less than 100 fg / TCID ₅₀ . In one embodiment, the high purity virus or high purity virus stock solution has a protein concentration of less than 60 fg / TCID ₅₀ . In another embodiment, the high purity virus or high purity virus stock solution has a DNA concentration of less than ₅₀ fg / TCID ₅₀ . In one embodiment, the high purity virus or high purity virus stock solution has a DNA concentration of less than 15 fg / TCID ₅₀ .

In a further embodiment, a high purity virus or high purity virus stock solution has a protein concentration and DNA concentration of less than 50 fg / TCID ₅₀ of less than 100 fg / TCID _50. In one embodiment, high purity virus or high purity virus stock solution has a protein concentration and DNA concentrations below 15fg / TCID ₅₀ of less than 60fg / TCID _50.

In a further embodiment, the high purity virus or high purity virus stock solution has a virus concentration of at least 10 ⁶ TCID ₅₀ / ml.

  A further embodiment of the invention is a method for evaluating and / or validating the virus clearance process and the materials used therein. A scaled virus clearance process is tested using the test sample and the results of the virus clearance process are analyzed. The amount of virus clearance evaluates the virus clearance process and the materials used in the virus clearance process and determines its validation. If the virus is sufficiently removed from the test sample to meet regulatory standards, the virus clearance process and materials used for clearance are validated.

In one embodiment, a method of evaluating and / or validating a virus clearance process includes the step of spiking a sample with a high purity virus stock solution to create a test sample. In one embodiment, high purity virus solution comprises a protein concentration of less than 100 fg / TCID _50, and a DNA concentration of less than 50 fg / TCID _50.

  Another embodiment of the present invention is a method for evaluating and / or validating a virus clearance process in a method comprising the step of spiking a sample with a virus solution, wherein the virus solution is flow-through chromatography, bead-based chromatography, size Prepared by methods that include exclusion treatment or treatment based on hydrophobicity. In one embodiment, the virus solution is prepared by cation exchange flow-through chromatography. In another embodiment, the virus solution is prepared by anion exchange flow-through chromatography. In one embodiment, flow-through chromatography includes cation exchange flow-through chromatography on a membrane or bead-based matrix.

In one embodiment, a method of evaluating and / or validate the materials used in the viral clearance process and there are at least ¹⁰ 7 _TCID 50 / ml virus, and protein concentration of less than 100 fg / TCID _50, less than 50 fg / TCID ₅₀ And spike the sample with a high purity virus stock solution comprising

That is, the gist of the present invention is as follows.
[1] In a method for preparing a high purity virus stock solution containing a protein concentration of less than 100 fg / TCID ₅₀ ,
(A) obtaining a virus sample prepared under low protein conditions;
(B) concentrating the virus sample in a suitable buffer;
(C) precipitating the virus in the virus sample, wherein the precipitated virus comprises a protein concentration of less than 100 fg / TCID ₅₀ ;
A step of producing a highly pure virus stock solution,
[2] The method according to [1], further comprising a step of treating the precipitated virus to remove trace amounts of impurities.
[3] The method according to [1], wherein the virus sample prepared under the low protein condition is a serum-free cell culture or a limited medium cell culture virus preparation,
[4] The method according to [1], wherein the step of concentrating the virus sample in a suitable buffer includes ultrafiltration, tangential flow filtration, dialysis, or a combination thereof.
[5] The method according to [1], wherein the suitable buffer is Tris-NaCl-EDTA (TNE),
[6] The method according to [1], wherein the step of precipitating the virus in the virus sample comprises ultracentrifugation, chemically induced precipitation, chromatographic binding, polymer induced aggregation, or a combination thereof.
[7] The method according to [6], wherein the chemically induced precipitation is polyethylene glycol (PEG) induced precipitation,
[8] The method according to [1], wherein the virus stock solution has a protein concentration of less than about 60 fg / TCID ₅₀ .
[9] The virus stock solution has a protein concentration and DNA concentrations below 15fg / TCID ₅₀ of less than 60fg / TCID _50, the method of [8],
[10] The method according to [2], wherein the further processing step includes flow-through chromatography, membrane adsorption, bead-based chromatography, size exclusion treatment, or treatment based on hydrophobicity,
[11] The method according to [10], wherein the further processing step includes cation exchange flow-through chromatography,
[12] The method according to [1], wherein the virus is a mammalian virus or bacteriophage, or a combination thereof,
[13] The method according to [1], wherein the virus is parvovirus or xenotropic murine leukemia virus (X-MuLV),
[14] The method according to [13], wherein the parvovirus is mouse minut virus (MVM).
[15] A virus stock solution prepared by the method according to [1],
[16] In a method for purifying a virus to a concentration of at least 10 ⁶ TCID ₅₀ / ml and a protein concentration of less than 100 fg / TCID ₅₀ ,
(A) obtaining a virus sample prepared under low protein conditions;
(B) concentrating the virus sample in a suitable buffer;
(C) precipitating the virus in the virus sample, the precipitated virus having a virus concentration of at least 10 ⁶ TCID ₅₀ / ml and a protein concentration of less than 100 fg / TCID ₅₀ ;
Comprising purifying the virus to a concentration of at least 10 ⁶ TCID ₅₀ / ml and a protein concentration of less than 100 fg / TCID ₅₀ ,
[17] The method according to [16], further comprising a step of treating the precipitated virus to remove trace amounts of impurities.
[18] The method according to [16], wherein the virus sample prepared under the low protein condition is a serum-free cell culture or a limited medium cell culture virus preparation,
[19] The method according to [16], wherein the step of concentrating the virus sample in a suitable buffer comprises ultrafiltration, tangential flow filtration, dialysis, or a combination thereof.
[20] The method according to [16], wherein the suitable buffer is Tris-NaCl-EDTA (TNE),
[21] The method according to [16], wherein the step of precipitating the virus in the virus sample comprises ultracentrifugation, chemically induced precipitation, chromatographic binding, polymer-induced aggregation, or a combination thereof.
[22] The method according to [21], wherein the chemically induced precipitation is polyethylene glycol (PEG) induced precipitation,
[23] The method according to [16], wherein the virus stock solution has a protein concentration of less than about 60 fg / TCID ₅₀ .
[24] The virus stock solution has a protein concentration and DNA concentrations below 15fg / TCID ₅₀ of less than 60fg / TCID _50, The method according to [23],
[25] The method according to [16], wherein the further processing step includes flow-through chromatography, membrane adsorption, bead-based chromatography, size exclusion processing, or treatment based on hydrophobicity,
[26] The method according to [25], wherein the further processing step includes cation exchange flow-through chromatography,
[27] The method according to [16], wherein the virus is a mammalian virus or bacteriophage, or a combination thereof,
[28] The method according to [16], wherein the virus is parvovirus or xenotropic murine leukemia virus (X-MuLV),
[29] The method according to [28], wherein the parvovirus is mouse minut virus (MVM),
[30] a virus purified by the method according to [16],
[31] In a method for evaluating a virus clearance process,
(A) Spiking a sample with a virus solution, the virus solution comprising: at least 10 ⁶ TCID ₅₀ / ml virus; a protein concentration less than 100 fg / TCID ₅₀ ; and a DNA concentration less than ₅₀ fg / TCID ₅₀ Producing a test sample thereby, and
(B) testing the scaled virus clearance process using the test sample;
(C) analyzing the results of the virus clearance process;
Including
A method wherein the amount of virus clearance determines the virus removal capacity of the virus clearance process, thereby evaluating the virus clearance process;
[32] The method according to [31], wherein the test sample contains at least 10 ⁵ TCID ₅₀ / ml of virus,
[33] The method of [31], wherein the virus clearance process comprises virus removal, virus inactivation, or a combination thereof,
[34] The virus removal method includes a filter and a chromatography medium, and the virus inactivation method includes low pH, high pH, ultraviolet (UV) irradiation, gamma irradiation, or temperature inactivation. [33] The method according to
[35] The method according to [31], wherein the virus is a mammalian virus or bacteriophage, or a combination thereof.
[36] The method according to [31], wherein the virus is parvovirus or xenotropic murine leukemia virus (X-MuLV),
[37] The method according to [36], wherein the parvovirus is mouse minut virus (MVM).
[38] In a method for evaluating a virus clearance process,
(A) spiking a sample with a virus solution, wherein the virus solution is prepared by a method comprising flow-through chromatography, thereby producing a test sample;
(B) testing the scaled virus clearance process using the test sample;
(C) analyzing the results of the virus clearance process;
Including
A method wherein the amount of virus clearance determines the virus removal capacity of the virus clearance process, thereby evaluating the virus clearance process;
[39] The method according to [38], wherein the flow-through chromatography comprises cation exchange flow-through chromatography on a membrane or bead-based matrix.

  The present invention provides a method for producing a high-titer and high-purity virus stock and a method for using the same.

2 is an overview of a general virus production protocol and an example of a specific mouse microvirus (MVM) production process. It is a flowchart which shows one Embodiment of this invention. 1 illustrates one embodiment of the present invention. It is a silver stained SDS-PAGE gel of the sample extract | collected at the various steps in a sample virus preparation method. Lanes 1, 14 and 15 are standard molecular weight markers. Lane 2 is a sample of MVM-infected cell culture medium on the third day immediately before the medium is changed from Dulbecco's modified Eagle medium (DMEM) containing 1% fetal bovine serum (FBS) to serum-free DMEM. Lane 3 is a sample of pooled cell lysate at the end of day 14 infection. Lane 4 is a clarified lysate sample. Lane 5 is a sample of the filtrate from ultrafiltration (Centricon®) that concentrates the virus. Lane 6 is a sample of diluted retentate from the ultrafiltration that concentrates the virus. Lane 7 is a sample of the supernatant after virus precipitation by ultracentrifugation. Lane 8 is a sample of virus pellet after virus precipitation by ultracentrifugation. Lane 9 is a sample of the virus pellet after virus precipitation by ultracentrifugation and filtration through a 0.22 micron filter. Lanes 10 and 12 are cations containing AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) crosslinked by MBAm (N, N′-methylenebisacrylamide) on a polyethersulfone (PES) matrix. A sample of the initial fraction of chromatographic polishing using exchange (CEX) membrane adsorbers. Lanes 11 and 13 are late fractions of chromatographic polishing using a CEX membrane adsorber. Source Willwand et al. J Virol (1993) 67: 5660, indicating that the molecular size of MVM capsid protein VP1 is 83 kDa and VP2 is 64 kDa. The two major protein bands revealed by silver staining SDS-PAGE (FIG. 3A) in the final purified virus product are the same size and ratio as the known MVM virus capsid proteins VP1 and VP2, and the major residual proteins are A very high purity virus stock is suggested which is a virus rather than an impurity. FIG. 3C is a table corresponding to the SDS-PAGE gel of FIG. 3A, with MVM virus titer (log TCID ₅₀ / ml), protein concentration (ug / ml and fg / TCID ₅₀ ), and DNA for each sample in lanes 2-13. Contains calculated values of concentration (ug / ml and fg / TCID50). 2 is a table demonstrating the reproducibility of the MVM fabrication process described herein. 1 illustrates one embodiment of the present invention. FIG. 5B is a table corresponding to the SDS-PAGE gel of FIG. 5B, with calculated values of MVM virus titer (log TCID ₅₀ / ml) and protein concentration (ug / ml and fg / TCID ₅₀ ) for each sample in lanes 2-11. Including. It is a silver stained SDS-PAGE gel of the sample extract | collected at the various steps in a sample virus preparation method. Lanes 1 and 12 are standard molecular weight markers. Lane 2 is a sample of MVM infected cell media on the third day just prior to switching from Advanced ™ DMEM with 1% FBS to serum-free Advanced ™ DMEM. Lane 3 is a sample of pooled cell lysate at the end of day 14 infection. Lanes 4 and 5 are clarified lysate samples. Lane 6 is a sample of the filtrate from ultrafiltration (Centricon®) that concentrates the virus. Lane 7 is a sample of the diluted retentate from the ultrafiltration that concentrates the virus. Lane 8 is a sample of the supernatant after virus precipitation by ultracentrifugation. Lane 9 is a sample of virus pellet resuspended after virus precipitation by ultracentrifugation. Lane 10 is a sample of the third fraction of chromatographic polishing using a CEX membrane adsorber. Lane 11 is a sample of the 27th fraction of chromatographic polishing using a CEX membrane adsorbent. 2 is a data table for high titer and high purity virus production using the method as described herein. FIG. 4 is a graph demonstrating the hydrodynamic performance of a test filter (Viresolve® Pro (V-Pro)), using a virus-spiked sample prepared as described herein, and a contract testing laboratory (CTO) compared to a sample spiked with a standard virus preparation typically used in validation studies. The ultracentrifugation method was used for virus preparation by CTO. MVM prepared as described herein spiked into a monoclonal antibody feed (9.1 g / L) that had previously shown filtration problems due to interaction with virus spikes. MVM made by CTO spiked to a concentration of 2 × 10 ⁵ TCID ₅₀ / ml, resulting in dramatic throughput through the filter V-Pro compared to the unspiked feed baseline (no virus). Declined. In contrast, MVMs prepared as described herein are hydrodynamic at a spike level of 5 × 10 ⁵ TCID ₅₀ / ml and also at a 10-fold higher spike leading to 6 × 10 ⁶ TCID ₅₀ / ml. There was no effect on performance. FIG. 3 is a graph evaluating three hydraulic CTO MVM spike stocks prepared by different methods and evaluating their hydrodynamic performance when spiked into a monoclonal antibody (Mab) feed and processed through Viresolve Pro. 3 is a photograph of an SDS-PAGE gel analysis of three standard CTO MVM preparations and an MVM preparation according to the method of the present invention. Proteins in 4-15% gradient gels were visualized by silver staining. Each sample lane was loaded with 10 μl of sample. Lane 1: molecular weight marker; Lane 2: CTO crude MVM preparation (2.3 × 10 ⁷ TCID ₅₀ / ml); Lane 3: CTO ultracentrifugated purified MVM preparation (1.8 × 10 ⁷ TCID ₅₀ / ml); Lane 4 (2.3 × 10 ⁷ TCID ₅₀ / ml): CTO Q membrane binding / elution purified MVM preparation; Lane 5: MVM preparation of the present invention (6.5 × 10 ⁸ TCID ₅₀ / ml); Lane 6 : Molecular weight marker.

  Contamination of biopharmaceuticals such as antibodies, recombinant proteins, vaccines, blood products, plasma, and animal products with bacteria, viruses, prions, etc. is a serious risk that requires adequate treatment. Contamination can occur in a variety of ways. For example, contamination can occur because raw materials (eg, cells in cell culture, blood products, etc.) are essentially contaminated with viruses. Biopharmaceutical manufacturing processes are also susceptible to virus contamination from exogenous sources (eg, inadvertent introduction through the use of non-sterile or inappropriately sterilized materials). Due to the nature of the product, biopharmaceutical manufacturers are highly regulated and obliged to incorporate sufficient virus clearance steps into their manufacturing process to ensure that the product is free of contaminants. The manufacturing process can incorporate multiple virus clearance steps. To be approved for a biopharmaceutical manufacturing process, each of these viral clearance steps must be evaluated for their effectiveness and thus validated. The virus clearance process typically includes either a virus removal process or a virus inactivation process.

  Virus removal is a method of physically removing virus from a sample. This is often achieved by either nanofiltration or chromatography. Nanofiltration techniques remove viruses by size exclusion. Whether the chromatographic method is successful in removing viruses depends on the column composition and reagents (eg, buffer) used.

  Virus inactivation is a method in which the virus can remain in the final product but remains in an inactive (inactive) form. Many viruses contain a lipid or protein coat that can be chemically altered to inactivate. Alternatively, the virus is completely denatured by a virus inactivation method. Examples of virus inactivation methods include solvent and / or detergent inactivation, pasturization (eg, heating to high temperature), pH inactivation (eg, using acidic pH), and radiation (For example, ultraviolet (UV) irradiation or γ-ray irradiation).

  Concentration of contaminating virus in the manufacturing process, i.e. the risk of virus contamination is very low, however, the virus is inherently infectious, so even a single virus particle will ruin all the run of the manufacturing process. Can be enough. For this reason, special measures must be taken to determine appropriate removal or inactivation methods in the manufacturing process. Therefore, it is necessary to evaluate and validate the effectiveness of such virus clearance methods. The spike test was created specifically for this purpose.

  The spike test uses a scale-down model of the virus clearance process of the production scale process to evaluate and / or validate the virus clearance process and to evaluate and / or validate the equipment used in the virus clearance method. The purity and titer of the virus stock affects how well the scale-down model reflects the production scale process. Impurities in the virus stock (eg DNA, RNA, proteins, lipids, non-infectious particles and virus aggregates) adversely affect the results of the spike test. For example, impurities can interfere with the action of chemicals that inactivate viruses, inhibit the effectiveness of radiation, and change the binding of target factors to chromatographic media (eg, lack of binding to chromatographic media). The virus clearance rate can be altered by being properly suppressed or by undesirably increasing binding) or by fouling (eg, plugging or clogging) the filter. As the fouling of a test unit (eg, filter or chromatographic media) is accelerated or its fouling characteristics change, the final fluid passing through the virus filter is reduced. As such impurities are usually not present in production scale units, the result is an underestimation of the actual throughput capability of the filter. For this reason, the apparent low performance of the unit in the validation test is compensated, and the production scale unit becomes excessive. Current industry standard virus stock production methods (eg, used in spike testing) generally produce virus stocks with high impurity content.

  In addition, a virus stock with the highest possible titer is also desirable. This is due in part to regulatory guidelines limiting the amount of virus spike that can be added to the test system. Thus, the titer of the virus stock determines the maximum possible concentration of virus spiked into the test material. As a result, the actual ability of the test unit to purify the virus in the production process can be underestimated. Current methods lack the ability to produce high purity and high titer virus stocks.

  The virus preparation methods described herein produce very high purity and high titer viruses. These clean virus stocks are designed to allow high concentration spikes in virus clearance tests without affecting the performance of scaled down unit operations. This is particularly useful for the evaluation and validation of small virus retention filters, such filters can be sensitive to virus stock impurities, especially when combined with certain feedstocks. In addition, the higher the virus titer, the higher the virus reduction rate can be demonstrated and the more severe challenges for virus clearance unit operation become possible.

  Exemplary viruses used in virus clearance tests that can be made with high titer and purity using the methods described herein include mammalian viruses and bacteriophages, such as And without limitation: RNA enveloped viruses (eg, human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), xenotropic, amphotropic, And ecotropic recombinant mouse retroviruses such as xenotropic murine leukemia virus (X-MuLV), retroviruses such as visna virus; flaviviruses such as bovine viral diarrhea virus (BVDV); vesicular stomatitis virus (VSV), etc. Rhabdoviruses; Paline Fluen Paramyxoviruses such as type 3 (PIV3)); RNA non-enveloped viruses (eg reoviruses such as reovirus type 3 (reo-3); picornaviruses such as poliovirus type 1 (PV-1); brain Myocarditis (EMC) virus and picornaviruses such as hepatitis A virus (HAV)); DNA enveloped viruses (eg, pseudorabies virus (PRV), bovine infectious rhinotracheitis (IBR) virus, and herpes simplex virus) Herpesviruses such as type 1 (HSV-1); poxviruses such as orf virus); DNA non-enveloped viruses (eg, adenoviruses such as human adenovirus; polyomaviruses such as simian virus 40 (SV40)), and swine Parvovirus (PPV), dog pal Parvoviruses such as virus (CPV), parvovirus B19, and murine microvirus (MVM) (also known as mouse minute virus, murine minute virus, MMV); and Phi Bacteriophages such as -X 174 (or Phi-X), Pseudomonas phage PP7, enterobacterial phage PR772, and Pseudomonas phage Phi-6.

  The described method has been successfully applied to mouse microvirus (MVM). Among the smallest and most difficult to remove or inactivate viruses, this parvovirus is the “gold standard” for virus clearance, especially virus filtration of biopharmaceuticals. Therefore, this method is also applicable to other viruses, particularly viruses similar in nature to MVM (eg, non-enveloped viruses produced by cells at relatively high titers). For example, porcine parvovirus (PPV), an important gold standard virus in the plasma industry, is structurally very similar to MVM.

  For practical reasons, the process steps that are evaluated and validated are scaled down. This is because it is not practical to use the actual production scale for the virus clearance test due to the amount of virus necessary for the virus spike test. Moreover, as will be readily apparent, it may be inappropriate to introduce infectious viruses into the manufacturing facilities of the current good manufacturing practice (cGMP) for testing purposes. Virus clearance is the rate of virus reduction (also referred to herein as “virus reduction rate” or “VRF”) by comparing the viral load of the spiked starting material to the treated material obtained from the test unit. It is calculated by calculating | requiring.

  The general production procedure for high purity and high titer virus, as well as the specific virus purification techniques used for MVM, are shown schematically in FIG.

  One embodiment of the present invention is a method of preparing a high purity virus or high purity virus stock solution. As used herein, a “high purity virus” is a virus that has a very low concentration of impurities (eg, proteins, DNA, lipids). In one embodiment, the high purity virus is less than 50 ug / ml, less than 45 ug / ml, less than 40 ug / ml, less than 35 ug / ml, less than 30 ug / ml, less than 25 ug / ml, less than 20 ug / ml, less than 15 ug / ml, It has a protein concentration of less than 10 ug / ml, less than 5 ug / ml, or less.

In another embodiment, the high purity virus or high purity virus stock solution is less than 100 fg / TCID _50, less than 90fg / TCID _50, less than 80fg / TCID _50, less than 70fg / TCID _50, less than 60fg / TCID _50, 50fg / TCID less than _50, less than 40fg / TCID _50, less than 30fg / TCID _50, less than 20fg / TCID _50, less than 10 fg / TCID _50, having less than 5 fg / TCID _50, or the following protein concentrations.

In a further embodiment, the high purity virus or high purity virus stock solution is less than ₅₀ fg / TCID _50, less than 45 fg / TCID _50, less than 40 fg / TCID _50, less than 35 fg / TCID _50, less than 30 fg / TCID ₅₀ , 25 fg / TCID ₅₀ Less than, 20 fg / TCID _50, less than 15 fg / TCID _50, less than 10 fg / TCID _50, less than 5 fg / TCID _50, less than 1 fg / TCID _50, less than 0.5 fg / TCID ₅₀ , or less.

In yet another embodiment, the high purity virus or high purity virus stock solution has a protein concentration as defined above and a DNA concentration as defined above. In one embodiment, high purity virus or high purity virus stock solution has a protein concentration and DNA concentration of less than 50 fg / TCID ₅₀ of less than 100 fg / TCID _50. In another embodiment, the high purity virus or high purity virus stock solution has a protein concentration and DNA concentrations below 15fg / TCID ₅₀ of less than 60fg / TCID _50. In a further embodiment, a high purity virus or high purity virus stock solution has a protein concentration and DNA concentrations below 0.5fg / TCID ₅₀ of less than 5 fg / TCID _50.

As one skilled in the art will appreciate, TCID ₅₀ / ml is a measure of the amount of a pathogenic factor that causes a pathological change in 50% of the cell culture inoculated with the pathogenic factor and is expressed as “tissue culture infectious dose. 50 "(TCID ₅₀ ). Methods for determining TCID ₅₀ values are known in the art. In general, serial dilutions of virus stock are prepared and inoculated into homotypic cell cultures, for example, in a multi-well format (eg, a 96-well plastic plate). The number of infected cell cultures is then determined by examining the cytopathic effect (CPE), typically for each virus dilution after the cell culture period. In a typical test, when the virus is serially diluted, none of the cell cultures are infected due to the absence of particles. When the virus is serially diluted at low fold, any cell culture is infected. When half of the cell culture shows a cytopathic effect with a specific serial dilution of virus, that is the dilution of virus that infects 50% of the cell culture. This number can be calculated from the data and is expressed as 50% tissue culture infectious dose per milliliter (TCID ₅₀ ).

In one embodiment, the virus stock is at least or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / Ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml It has a virus concentration of 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or higher.

  The method comprises the steps of obtaining a virus sample prepared under low protein conditions, concentrating the virus sample, exchanging with a suitable buffer, and precipitating the virus in the virus sample. The virus thus precipitated is a high purity virus. In one embodiment, the high purity virus has a viral titer as described above.

  Viral samples prepared under low protein conditions can be achieved using any suitable method. For example, virus samples can be obtained from virus-infected cells cultured in low serum medium culture, serum-free medium culture, limited medium culture, or a combination thereof. In one embodiment, the cells are infected with low serum-containing cell culture medium (eg, Dulbecco's Modified Eagle Medium (DMEM) containing 1% fetal bovine serum (FBS)) and then serum-free cell culture medium (eg, FBS-free). In DMEM-containing or FBS-free Advanced DMEM) or limited serum-free cell culture medium. In another embodiment, the cells are infected with serum-free cell culture medium (eg, FBS-free DMEM, or FBS-free Advanced DMEM), or limited serum-free cell culture medium.

The virus is concentrated and the cell culture medium is replaced with a suitable buffer. For example, the virus may be in a suitable buffer, such as Tris-NaCl-EDTA (TNE) buffer (eg, 10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA); phosphate buffered saline (PBS). Buffer (eg, 137 mM NaCl, 2.7 mM KCl, 100 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4); Tris buffered saline (TBS) (eg, 50 mM Tris, 150 mM) NaCl, pH pH 7.6); Tris-EDTA (TE) buffer (eg, 10 mM Tris, 1 mM EDTA); or 10 mM Tris.

  In one embodiment, the viral sample is concentrated by ultrafiltration (eg, ultrafiltration centrifuge), tangential flow filtration, dialysis, or a combination thereof. In ultrafiltration, a sample is processed through a membrane that passes fluids and small molecules (eg, salts, sugars, and proteins whose diameter is smaller than the membrane pore size) but retains the virus. The fluid can be forced through the ultrafiltration membrane by a number of methods including, but not limited to, centrifugation, peristaltic pumps, vacuum and air pressure. The virus is then resuspended in the desired amount of fresh buffer. In tangential flow filtration (also known as cross flow filtration), the feed solution flows perpendicular to the surface of the ultrafiltration membrane. While the virus does not pass, a transmembrane differential pressure is created that allows fluids and small molecules to flow through the membrane, reducing the volume in the sample. The buffer can then be replaced by supplying fresh buffer to the tangential flow filtration system while continuing to draw fluid through the membrane. In dialysis, the sample is placed in a bag-like membrane that allows fluids and small molecules to pass freely but keeps the virus inside. By placing this “dialysis bag” in a large volume of buffer solution at the specified osmolarity, the cell culture medium in the sample is diluted in a new buffer solution, and the volume in the bag is reduced, so that the buffer solution is replaced. And virus concentration is achieved. In a detailed embodiment, the virus sample is concentrated by ultrafiltration and buffer exchange to TNE.

  Precipitation of the virus in the virus sample can be achieved by any suitable method. For example, ultracentrifugation, chemically induced precipitation, chromatographic binding, polymer induced aggregation, or combinations thereof. Ultracentrifugation techniques are standard in the art. For example, a viral sample can be ultracentrifuged at about 4 ° C. and at least about 25,000 RPM (112600 × g) for at least about 4 hours using standard equipment and collected as a pellet at the bottom of the tube. Samples may be added after placing a “cushion” (eg glycerol) of high density media at the bottom of the tube, so that the virus must pass through this high density layer to reach the pellet at the bottom of the tube. On the other hand, no impurities pass through. The virus can also be centrifuged through a density gradient medium, such as cesium chloride or iodixanol, and the virus collected as a concentrated layer at some point along the density gradient. Virus precipitation using chemical agents can be any agent that binds the virus and forms insoluble aggregates (eg, any suitable molecular weight (MW), eg, about MW 6,000 to about MW 10, 000 polyethylene glycol (PEG)). Chromatographic binding can be achieved using chromatographic media suitable for binding virus (eg, Q-type or S-type chemistry with agarose, controlled pore glass or membrane media). Soluble polymers that agglutinate inductively (eg, smart polymers such as polyethylene, polyvinyl chloride, polystyrene, polypropylene, and polyvinyl alcohol) can also be used to precipitate viruses in the virus sample. In one embodiment, the virus is precipitated by ultracentrifugation.

  In one embodiment, the method further comprises chromatographic polishing of the precipitated virus. As used herein, “chromatographic polishing” is a method of passing traces of a sample through a medium that passes most of the virus while retaining impurities based on their electrostatic charge or hydrophobicity. It means removing impurities. Chromatographic polishing typically removes impurities that are not removed by virus precipitation. In one embodiment, the virus is subjected to flow-through chromatography polishing (eg, using a cation exchange (CEX) membrane adsorber, or an anion exchange medium such as ChromaSorb ™). Such chromatographic polishing removes impurities such as proteins, nucleic acids, lipids, and sugars from a virus sample based on their electrostatic charge. Other chromatographic polishing methods such as other membrane adsorbents (eg Pall Mustang S); bead-based chromatographic media with appropriate chemistry (S or Q depending on virus and buffer conditions); size exclusion media; Hydrophobic-based media) can be used. As one skilled in the art will appreciate, the choice of chromatographic polishing apparatus used will depend on the nature of the virus to be purified, the contaminants to be removed, and the scale of purification.

  In one embodiment of the method described herein, the virus flows through the chromatographic polishing medium without binding, while the impurities bind to the medium and are thus removed from the sample. Buffer conditions may be adjusted (eg, changing salt content and / or pH values) to maximize virus passage and impurity removal. In one embodiment, the MVM in TNE buffer, pH 7 is passed through a cation exchange (CEX) membrane adsorber to adsorb and remove protein and DNA impurities while the majority of the virus passes through. To do.

  In another embodiment, chromatographic polishing is applied to any virus preparation and purification method for purifying virus to produce high purity virus. For example, in addition to those described herein, other virus preparation and purification methods that can be further purified by chromatographic polishing include viruses prepared by ultracentrifugation pellet formation, gradient purification, and ultrafiltration. . Chromatographic polishing can also be used for virus purification from unpurified virus preparations that have only been processed by clarification to remove cell debris. Such virus preparations may be purified in a flow-through manner, using, for example, chromatographic media appropriate to the specific virus used, buffer conditions, and the scale of the method. In one embodiment, virus (eg, MVM) concentrated by ultracentrifugation pellet formation is passed through a cation exchange (CEX) membrane. In one embodiment, the CEX membrane comprises AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) crosslinked by MBAm (N, N′-methylenebisacrylamide) on a polyethersulfone (PES) matrix. Including. Virus (eg, MVM) passes through the membrane without significant binding, while impurities such as proteins and DNA bind to the membrane and are removed from the virus sample.

  The methods described herein greatly reduce the presence of impurities in the virus stock. For example, the total amount of impurities in the virus stock is greatly reduced. “Total amount of impurities” refers to a decrease in the amount of impurities (eg, protein, DNA as determined by standard assays such as bicinchoninic acid (BCA) assay for protein quantification, PicoGreen ™ for DNA quantification, etc. The number of micrograms or femtograms of impurities such as is reduced by about 90 to about 99%). In addition, the number of impurities is greatly reduced. For example, protein impurities that can be identified on a protein gel are only slightly in contrast to hundreds, thousands, or tens of thousands of different proteins as determined by methods such as silver staining of protein SDS-PAGE gels. There may be several (eg, 2, 3, 5, 10, or 20) different proteins. Impurities are contaminants of virus preparations such as DNA, RNA, proteins, lipids, non-infectious particles and virus aggregates.

In one embodiment, the virus stock is at least or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / Ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml Virus concentrations of 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or higher, and 100 ug / ml, 90 ug / ml, 80 ug / ml, 70 ug / ml, 60 ug / ml, 50 ug / ml, 40 ug / ml, 30 ug / ml, 20 u Has a protein concentration of protein lower than or nearly lower than g / ml, 10 ug / ml or lower.

In one embodiment, the virus stock is at least or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / Ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml Virus concentration of 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or higher, and 100 fg / TCID ₅₀ , 90 fg / TCID ₅₀ , 80 fg / TCID ₅₀ , 70 fg _{/ TCID 50, 60fg / TCID 50} , 50f _{/ TCID 50, 40fg / TCID 50} , 30fg / TCID 50, 20fg / TCID 50, 15fg / TCID 50, 10fg / TCID 50, 5fg / TCID 50, or less than or less, or has a substantially lower protein concentrations than that.

In one embodiment, the virus stock is at least or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / Ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml Virus concentration of 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or higher, and 30 fg / TCID ₅₀ , 25 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg _{/ TCID 50, 10fg / TCID 50} , 5fg / _{CID 50, 1fg / TCID 50,} 0.5fg / TCID 50, or less than or less, or has a substantially lower DNA concentrations than that.

In one embodiment, the virus stock is at least or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / Ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml Virus concentrations of 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or higher; 100 fg / TCID ₅₀ , 90 fg / TCID ₅₀ , 80 fg / TCID ₅₀ , 70 fg / _{TCID 50, 60fg / TCID 50,} 50fg / _{CID 50, 40fg / TCID 50,} 30fg / TCID 50, 20fg / TCID 50, 15fg / TCID 50, 10fg / TCID 50, 5fg / TCID 50, or less than or less, or substantially lower than the protein concentration; and 30Fg / TCID ₅₀ , 25 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID ₅₀ , 5 fg / TCID ₅₀ , 1 fg / TCID ₅₀ , 0.5 fg / TCID ₅₀ , or lower, or substantially lower Has a low DNA concentration.

As those skilled in the art will appreciate, by applying the methods described herein, many viruses can be at least, or at least approximately 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 10 ^8. It can be purified to a concentration of TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml or higher. Thus, in one embodiment, the sequential steps of obtaining a virus sample prepared under low protein conditions, concentrating the virus sample in a suitable buffer, and precipitating the virus in the virus sample are performed. including a method of purifying virus concentration of at least ¹⁰ 6 _TCID 50 / ml, wherein the precipitated virus has a viral concentration of at least ¹⁰ 7 _TCID 50 / ml, virus thus at least ¹⁰ 7 _TCID 50 / ml Purified to a concentration of

In one embodiment, the method comprises at least or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID. ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml Purify to a concentration of ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or higher.

In another embodiment, the method at least, or at least about 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / Ml, 5 × 10 ¹¹ TCID ₅₀ / ml or higher, and 100 ug / ml, 90 ug / ml, 80 ug / ml, 70 ug / ml, 60 ug / ml, 50 ug / ml, 40 ug / ml, 30 ug / ml , 20 ug / ml, or lower, or nearly lower protein concentration To purify the protein.

In one embodiment, the method comprises at least or at least approximately 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID. ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml ml, 5 × 10 ¹¹ TCID ₅₀ / ml or higher, and 100 fg / TCID ₅₀ , 90 fg / TCID ₅₀ , 80 fg / TCID ₅₀ , 70 fg / TCID ₅₀ , 60 fg / TCID ₅₀ , ₅₀ fg / TCID ₅₀ , 40 fg / TCID ₅₀ , 30 fg / TCID ₅₀ , _{20fg / TCID 50, 15fg / TCID} 50, 10fg / TCID 50, 5fg / TCID 50, or less than or less, or purified substantially lower protein concentrations than that.

In one embodiment, the method comprises at least or at least approximately 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID. ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml ml, 5 × 10 ¹¹ TCID ₅₀ / ml or higher, and 30 fg / TCID ₅₀ , 25 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID ₅₀ , 5 fg / TCID ₅₀ , 1 fg / TCID ₅₀ , 0.5 fg / TCID ₅₀ , or Purify to lower or nearly lower DNA concentrations.

In one embodiment, the method comprises at least or at least approximately 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID. ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml ml, 5 × 10 ¹¹ TCID ₅₀ / ml or higher concentration; 100 fg / TCID ₅₀ , 90 fg / TCID ₅₀ , 80 fg / TCID ₅₀ , 70 fg / TCID ₅₀ , 60 fg / TCID ₅₀ , ₅₀ fg / TCID ₅₀ , 40 fg / TCID ₅₀ , 30 fg / TCID ₅₀ , 20 _{fg / TCID 50, 15fg / TCID} 50, 10fg / TCID 50, 5fg / TCID 50 or less than or less, or substantially lower protein concentrations than that; and _{30fg / TCID 50, 25fg / TCID} 50, 20fg / TCID 50, Purify to a DNA concentration lower than, or lower than, 15 fg / TCID ₅₀ , 10 fg / TCID ₅₀ , 5 fg / TCID ₅₀ , 1 fg / TCID ₅₀ , 0.5 fg / TCID ₅₀ , or lower.

  Another embodiment of the present invention is a high purity virus or high purity virus stock produced by the methods described herein. In one embodiment, a composition comprising, consisting essentially of, or consisting of a high purity virus or high purity virus stock made by the methods described herein.

  Further embodiments of the present invention include a viral clearance process and the operation of the viral clearance unit used in the clearance process (eg, a flat membrane or hollow fiber run filter of either direct flow or tangential flow mode; Chromatographic processes including ion exchange, anion exchange, hydrophobic exchange, mixed chromatography, affinity chromatography such as protein A or other ligand-based adsorption; low pH, solvent / surfactant, radiation (gamma radiation) Or UV), virus inactivation methods such as heat treatment) and / or validate methods.

  As discussed above, one method for assessing and / or validating the virus clearance process is the virus spike test in a scaled-down virus clearance process model (also referred to herein as the “scaled virus clearance process”). is there. The purpose of this virus spike test is to evaluate the effectiveness of the virus clearance unit in virus purification from the manufacturing process of products (such as biopharmaceuticals). Higher virus titers can demonstrate higher virus reduction rates and allow for more demanding challenges for virus clearance unit operation. However, in spike testing, the spike material can be kept in small amounts, typically less than 10% (vol / vol) relative to the sample under test, so that the composition of the sample product does not unacceptably change. desirable. Therefore, it is desirable to use a virus spike with a high virus titer. The sample used in validation or evaluation of the virus clearance process and / or virus clearance unit used in the virus clearance process is any suitable manufacturing process feed sample. For example, a suitable sample is representative of a product made in biopharmaceutical manufacturing. Examples include sample solutions containing antibodies, recombinant proteins, vaccines, conjugate proteins, blood products, plasma, livestock products and the like.

Thus, one embodiment is a method for evaluating and / or validating a virus clearance process, the method comprising the step of spikeing a sample with a high purity virus solution. In one embodiment, high purity virus solution to produce a test sample having a protein concentration and DNA concentration of less than 50 fg / TCID ₅₀ of less than 100 fg / TCID _50. In one embodiment, the high purity virus stock solution has high purity virus at a virus concentration of at least, or at least about 10 ⁶ TCID ₅₀ / ml to at least, or at least about 10 ⁷ TCID ₅₀ / ml, or higher. A scaled virus clearance process, a scaled-down virus clearance process model, is tested using test samples, the results of the virus clearance process are analyzed, and thus the virus clearance process is evaluated by the amount of virus clearance, and Its validation is determined.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating the virus clearance process is 100 ug / ml, 90 ug / ml, 80 ug / ml, 70 ug / ml, 60 ug / ml, 50 ug / ml, Contains protein at a protein concentration lower than or nearly lower than 40 ug / ml, 30 ug / ml, 20 ug / ml, or less. In another embodiment, the high purity virus solution used in the method for evaluating and / or validating the virus clearance process is 100 fg / TCID ₅₀ , 90 fg / TCID ₅₀ , 80 fg / TCID ₅₀ , 70 fg / TCID ₅₀ , 60 fg / TCID. ₅₀ , ₅₀ fg / TCID ₅₀ , 40 fg / TCID ₅₀ , 30 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID ₅₀ , 5 fg / TCID ₅₀ , or lower, or substantially lower protein concentration including. In a further embodiment, the high purity virus solution used in the method of evaluating and / or validating the virus clearance process is 30 fg / TCID ₅₀ , 25 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID _50. It includes a DNA concentration lower than or nearly lower than ₅ fg / TCID ₅₀ , 1 fg / TCID ₅₀ , 0.5 fg / TCID ₅₀ , or lower. In yet another embodiment, the high purity virus solution used in the method of evaluating and / or validating the virus clearance process is at least, or at least about 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more.

In another embodiment, the high purity virus solution used in the method of assessing and / or validating the virus clearance process is at least, or at least about 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ^6. TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / Ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more viruses, And 100 ug / ml, 90 ug / ml, 80 ug / ml, 70 ug / m 1, 60 ug / ml, 50 ug / ml, 40 ug / ml, 30 ug / ml, 20 ug / ml, or less, or less protein protein concentration.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating the virus clearance process is at least, or at least about 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID. ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more viruses, and _{100fg / TCID 50, 90fg / TCID} 50, 80fg / _{CID 50, 70fg / TCID 50,} 60fg / TCID 50, 50fg / TCID 50, 40fg / TCID 50, 30fg / TCID 50, 20fg / TCID 50, 15fg / TCID 50, 10fg / TCID 50, 5fg / TCID 50, or Includes protein concentrations below or nearly below.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating the virus clearance process is at least, or at least about 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID. ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more viruses, and _{30fg / TCID 50, 25fg / TCID} 50, 20fg / T _{ID 50, 15fg / TCID 50,} 10fg / TCID 50, 5fg / TCID 50, 1fg / TCID 50, 0.5fg / TCID 50, or less than or less, or a substantially lower DNA concentrations than that.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating the virus clearance process is at least, or at least about 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID. ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more virus; 100 fg _{/ TCID 50, 90fg / TCID 50} , 80fg / TC _{D 50, 70fg / TCID 50,} 60fg / TCID 50, 50fg / TCID 50, 40fg / TCID 50, 30fg / TCID 50, 20fg / TCID 50, 15fg / TCID 50, 10fg / TCID 50, 5fg / TCID 50, or A protein concentration below or nearly below; and below 30 fg / TCID ₅₀ , 25 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID ₅₀ , 5 fg / TCID ₅₀ , or below, or Contains approximately lower DNA concentrations.

In one embodiment of the method for assessing and / or validating a virus clearance process, the test sample is at least or at least approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001% (vol / vol) spiked with any one of the above high purity virus solutions. In another embodiment of the method of assessing and / or validating the virus clearance process, the test sample is at least, or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × from any one of the high purity virus solutions described above. 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ Spiked with virus / ml or more.

  If the virus is sufficiently removed from the test sample to meet regulatory standards, those data can be applied to virus clearance process validation. In one embodiment, the scaled virus clearance process includes a filter.

A further embodiment is a method for evaluating and / or validating a viral clearance unit. The method includes the step of spiking the sample with a high purity virus solution as described above. In one embodiment, high purity virus to produce a test sample contains a protein concentration of less than 100 fg / TCID _50, and a DNA concentration of less than 50 fg / TCID _50. In another embodiment, the high purity virus solution comprises at least, or at least about 10 ⁶ TCID ₅₀ / ml virus to at least, or at least about 10 ⁷ TCID ₅₀ / ml virus, or more. The virus clearance unit, which is a scale-down model of the virus clearance unit used in the virus clearance process, is tested using test samples, the results of the virus clearance unit are analyzed, and thus the effectiveness of the virus clearance unit is determined by the virus. Determined by the amount of clearance.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating virus clearance units is 100 ug / ml, 90 ug / ml, 80 ug / ml, 70 ug / ml, 60 ug / ml, 50 ug / ml, Contains protein at a protein concentration lower than or nearly lower than 40 ug / ml, 30 ug / ml, 20 ug / ml, or less. In another embodiment, the high purity virus solution used in the method of evaluating and / or validating virus clearance units is 100 fg / TCID ₅₀ , 90 fg / TCID ₅₀ , 80 fg / TCID ₅₀ , 70 fg / TCID ₅₀ , 60 fg / TCID. ₅₀ , ₅₀ fg / TCID ₅₀ , 40 fg / TCID ₅₀ , 30 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID ₅₀ , 5 fg / TCID ₅₀ , or lower, or substantially lower protein concentration including. In a further embodiment, the high purity virus solution used in the method of evaluating and / or validating virus clearance units is 30 fg / TCID ₅₀ , 25 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID _50. It includes a DNA concentration lower than or nearly lower than ₅ fg / TCID ₅₀ , 1 fg / TCID ₅₀ , 0.5 fg / TCID ₅₀ , or lower. In yet another embodiment, the high purity virus solution used in the method of assessing and / or validating a virus clearance unit is at least, or at least about 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, or more.

In another embodiment, the high purity virus solution used in the method of evaluating and / or validating virus clearance units is at least, or at least about 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ^6. TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / Ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more viruses, And 100 ug / ml, 90 ug / ml, 80 ug / ml, 70 ug / m 1, 60 ug / ml, 50 ug / ml, 40 ug / ml, 30 ug / ml, 20 ug / ml, or less, or less protein protein concentration.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating a virus clearance unit is at least, or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID. ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more viruses, and _{100fg / TCID 50, 90fg / TCID} 50, 80fg / _{CID 50, 70fg / TCID 50,} 60fg / TCID 50, 50fg / TCID 50, 40fg / TCID 50, 30fg / TCID 50, 20fg / TCID 50, 15fg / TCID 50, 10fg / TCID 50, 5fg / TCID 50, or Includes protein concentrations below or nearly below.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating a virus clearance unit is at least, or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID. ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more viruses, and _{30fg / TCID 50, 25fg / TCID} 50, 20fg / T _{ID 50, 15fg / TCID 50,} 10fg / TCID 50, 5fg / TCID 50, 1fg / TCID 50, 0.5fg / TCID 50, or less than or less, or a substantially lower DNA concentrations than that.

In one embodiment, the high purity virus solution used in the method of evaluating and / or validating a virus clearance unit is at least, or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID. ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ / ml, or more virus; 100 fg _{/ TCID 50, 90fg / TCID 50} , 80fg / TC _{D 50, 70fg / TCID 50,} 60fg / TCID 50, 50fg / TCID 50, 40fg / TCID 50, 30fg / TCID 50, 20fg / TCID 50, 15fg / TCID 50, 10fg / TCID 50, 5fg / TCID 50, or Protein concentrations below or nearly below; and 30 fg / TCID ₅₀ , 25 fg / TCID ₅₀ , 20 fg / TCID ₅₀ , 15 fg / TCID ₅₀ , 10 fg / TCID ₅₀ , 5 fg / TCID ₅₀ , 1 fg / TCID ₅₀ ,. It contains a DNA concentration lower than or nearly lower than ₅ fg / TCID ₅₀ or lower.

In one embodiment of the method of assessing and / or validating viral clearance units, the test sample is at least or at least approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, Spike with any one of the above 2% or 1% (vol / vol) high purity virus solutions. In another embodiment of the method of assessing and / or validating viral clearance units, the test sample is at least, or at least approximately 10 ⁵ TCID ₅₀ / ml, 5 × from any one of the high purity virus solutions described above. 10 ⁵ TCID ₅₀ / ml, 10 ⁶ TCID ₅₀ / ml, 5 × 10 ⁶ TCID ₅₀ / ml, 10 ⁷ TCID ₅₀ / ml, 5 × 10 ⁷ TCID ₅₀ / ml, 10 ⁸ TCID ₅₀ / ml, 5 × 10 ⁸ TCID ₅₀ / ml, 10 ⁹ TCID ₅₀ / ml, 5 × 10 ⁹ TCID ₅₀ / ml, 10 ¹⁰ TCID ₅₀ / ml, 5 × 10 ¹⁰ TCID ₅₀ / ml, 10 ¹¹ TCID ₅₀ / ml, 5 × 10 ¹¹ TCID ₅₀ Spiked with virus / ml or more.

  A virus clearance unit is validated if the virus is sufficiently removed from the test sample to meet regulatory standards. In one embodiment, the virus clearance unit includes a filter.

  Virus removal achieved with a comprehensive scaled-down virus clearance test can be added to the virus clearance declaration reported at the time of regulatory filing of the drug application.

Example 1: MVM proliferation with 324K cells (see Figures 2-4)
material:
Proliferating cell line: 324K cells (human fibroblasts transformed with simian virus 40), freshly thawed from stock.
Infection: Murine minut virus (MVM) obtained from ATCC (Cat. No. VR-1346) virus stock was used to infect 324K host cells.
Medium: DMEM containing 10% fetal bovine serum “FBS”, DMEM containing 1% FBS, and DMEM containing no FBS. All were supplemented with 0.1 mM non-essential amino acids, 2 mL L-glutamine, 0.2 units / ml penicillin / streptomycin. FBS-free DMEM was supplemented with 0.1 mM non-essential amino acids, 2 mL L-glutamine, 0.1 units / ml penicillin / streptomycin.
Resuspension buffer: TNE (10 mM Tris [pH 8.0], 150 mM NaCl, 1 mM EDTA).
Apparatus: Millipore cation exchange (CEX) containing AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) crosslinked by MBAm (N, N′-methylenebisacrylamide) on a polyethersulfone (PES) matrix. ) Membrane adsorber, Millipore Centricon®-70 100 kDa centrifugal ultrafiltration device, and Millipore Millex ™ Durapore 0.22 μm filter.

Method:
The assay was performed at multiple stages within the virus stock preparation method described herein. In this example, the assay was performed at the points shown in red in FIG. The assay panel for each sample was as follows: virus titer (see below), BCA protein content (none for samples prior to Centricon®), PicoGreen ™ DNA content, and SDS. -PAGE silver stain. At each sample point, a 1 ml sample was taken for the assay.

Viral titration: Unless otherwise noted, 50 μl of sample was diluted to 10 ⁻² for each titration (ie diluted to 4.95 ml of medium). In exceptional cases the Centricon® filtrate was assayed starting from the undiluted sample. All samples were stored at 4 ° C. until titration, and then stored at −80 ° C. after titration.

  324K host cell line growth: From a freshly thawed working stock vial, 30 T150 flasks of 324K cells were grown to confluence. Growth medium: DMEM containing 10% FBS.

Growth and infection schedule: Day 0: 324K infection-detach 324K cells using trypsin. Suspended cells are pooled and pelleted at 1500 RPM for 10 minutes. Decant and discard the medium. Wash cells by resuspending in 50 ml of complete DMEM containing 1% FBS. Pellet cells as above. Decant and discard the medium. Pool the resuspended cells in a single sterile container and count the cells. The resulting cell count was 2.02E + 06 cells / ml for a total of 60.0 ml. The total number of cells was 1.2E + 08 cells. Therefore, the number of cells per confluent T150 was 9.2E + 06 cells. A cell volume of 650 ml was prepared at a density of 2.0 × 10 ⁵ cells / ml in DMEM containing 1% FBS. Specifically, 60 ml of cell suspension was added to 590 ml of 1% FBS-containing DMEM (total volume = 650 ml). Cell density count: 2.7E + 05 cells / ml. MVM was added directly to the suspended cells at a MOI of 0.002 TCID ₅₀ / cell. This requires adding ₅₀₀ TCID ₅₀ x 650 ml per ml of cells = adding a total of 3.3 x 10 ⁵ TCID ₅₀ . MVM stock titer: 1 × 10 ⁸ TCID ₅₀ / ml. 3.3 × 10 ⁵ TCID ₅₀ ÷ stock titer = 650 ml cells were added with 0.0033 ml = 3.3 μl of MVM stock (33 μl of 1/10 dilution stock may be added). The cell suspension was mixed thoroughly. 20 ml of the cell / virus suspension was transferred to 30 T150 flasks (named flasks 1-30). Target seeding = 4.0 × 10 ⁶ cells / T150 flask. Actual seeding 5.4E6 cells / T150. Cells were incubated at 37 ° C., 10% CO ₂ . Day 3-4: Replace with serum-free medium. Change the medium in half of the flask on each of days 3-4: On day 3, change the medium in flasks 1-15; on day 4, change the medium in flasks 15-30.

  Medium exchange procedure: Decant the medium in each flask into a common pool. One pool is created per collection per day: medium d3 m / c from flasks 1-15 (day 3 medium change); medium d4 m / c from flasks 15-30. Assay each decanted media pool. Wash each flask with 20 ml DMEM [no FBS]. Pour and discard the wash medium. The medium is replaced with 20 ml DMEM [no FBS] and then cell incubation is continued.

  About day 14: Collect lysate. When the cells show complete CPE (virus-induced lysis), the cell lysate is recovered by decanting the flask into three pools. Lysate d3 m / c; lysate d4 m / c. Each lysate pool is assayed (virus titer only). Add lysate to a single pool. The combined lysate pool is assayed (virus titer only). The lysate is stored at 4 ° C. until further processed.

  Clarification: Centrifuge the lysate by spinning at 2000 xg for 15 minutes. Decant and save supernatant (discard pellet). Filter the supernatant with 0.22 μm GP Express Plus (PVDF) Stericup. Total volume of clarified lysate: 532.6 ml (determined by comparing the weight of empty and full Stericup). Assay the clarified lysate.

  Concentration with Centricon®: Precool the centrifuge to 4 ° C. Four Centricon® Plus 100 kDa devices are loaded with 70 ml of each clarified lysate = total 280 ml. Spin Centricon® tube to retentate volume = 1-2 ml (Thermo Fisher tabletop centrifuge, rotor: 228, temperature: 4 ° C., speed: 2390 × g, total spin time (spin 1): 13 minutes) . Store the filtrate in a common filtrate pool. Refill Centricon® tubes with more than about 70 ml lysate per device (inject above the previous retentate). Spin Centricon® tube again as above to retentate volume = 1-2 ml. Total spin time (spin 2): 16.45 minutes. Add the filtrate to the previous filtrate from the previous spin. The Centricon® filtrate is assayed (which detects any virus leaks and visualizes / quantifies the proteins and DNA removed by the Centricon® process). Remove any remaining clarified lysate. Wash the Centricon® tube by filling approximately 70 ml of TNE. Spin Centricon® tube again as above to retentate volume = 1-2 ml. Total spin time (spin 3-wash): 13 minutes). Discard the washing filtrate. The apparatus core is inverted and the retentate is collected by centrifugation (rotor: 228, temperature: 4 ° C., speed: 1000 × g, time: 2 minutes). Mix the retentate in a common pool (to be 31 mL, so a 1 mL sample can be taken).

  Ultracentrifugation: Weigh a 30 ml ultracentrifuge tube with a cap. Empty tube weight: 86.638 g. Use TNE to bring the retentate pool volume to 31 ml (thus removing the 1 ml for assay and capping will fill the ultracentrifuge tube). Assay the diluted retentate. The expected virus concentration rate at this point is 18X from the clarified lysate. Transfer the diluted retentate pool to a 30 ml ultracentrifuge tube. Add TNE if necessary to completely fill the tube. Weigh a full ultracentrifuge tube with a cap on it. Full tube weight: 115.692 g. Total diluted retentate volume (full-empty weight): 29.054 ml. Precool the ultracentrifuge to 4 ° C. Make a balance tube with the same weight as the virus tube. The virus is ultracentrifuged (ultracentrifugal rotor: SW28, temperature: 4 ° C., speed: 25,000 RPM (112600 × g), time: 4 hours). Carefully decant the supernatant from the tube. The supernatant is assayed. The pellet is resuspended in 15 ml TNE by pipetting up and down. Collect the resuspended virus in a new tube. Rinse the ultracentrifuge tube with another 15 ml TNE by pipetting up and down. Rinse solution is added to the initial pellet resuspension to form a 30 ml virus pool. Assay the resuspended pellet pool.

  Sterile filtration: Sterilize the filter using a 0.22 μm Durapore Millex syringe filter. The filtrate after 0.22 μm is assayed.

  CEX membrane adsorption treatment: 30 ml of virus pool is passed through a CEX membrane adsorption device. CEX procedure: CEX apparatus is pre-wetted with MilliQ for 10 minutes at 30 psi. Repeat the pre-wetting of the device with buffer. After pre-wetting, the device is removed from the pressure source and a hydrophobic Millex is attached to the V-Shield vent. Use a sterile syringe to slowly expel the remaining volume in the CEX device. Care should be taken not to apply full pressure to the device and some liquid should remain between the membrane layers. Using a syringe pump, set the syringe diameter and flow rate (2 mL / min). Load the syringe with the virus preparation and remove any air in the syringe. A pre-wetted CEX is attached to the syringe and secured to the syringe pump holder. Using tubing attached to the outlet side of the CEX device, start the syringe pump and collect the filtrate in a sterile container. After all material is pushed through the CEX, the pump is stopped, the syringe is removed, and the volume in the tubing is drained into the collection container. CEX filtrate is collected as 1 ml aliquots numbered 1-14 in 2 ml cyrovials. CEX-treated virus fractions 2 (early fraction) and 14 (late fraction) are assayed.

  Virus storage: snap freeze by immersing the vial in an isopropanol / dry ice bath. Immediately transfer the sample to a −80 ° C. storage room.

  The virus sample is thawed and examined for aggregation by filter sizing: Thaw an aliquot of virus in a 37 ° C water bath. Assay thawed virus. Each thawed aliquot is passed through a 0.1 μm syringe filter. Assay virus filtered through 0.1 μm filter.

Conclusion 324K cells produced approximately 7.7 log TCID ₅₀ / ml in an untreated lysate. Removal of serum did not reduce the titer from what was previously observed with FBS-containing preparations. Although Centricon® was able to recover most of the virus, no contaminating protein was removed. Centrifugation recovered most of the remaining virus, resulting in a remarkably high purity. Passing the resuspended pellet through a Millipore CEX membrane adsorber removed most of the remaining contaminants. Consistent results were observed for evaluation runs 1 to 4, all performed using this method.

Example 2: MVM proliferation with A9 cells (see FIGS. 5-7)
The method of Example 1 was repeated using A9 cells (ATCC) instead of 324K host cells and Advanced ™ DMEM (Invitrogen product number 12491-015) instead of DMEM. Fractions after CEX were collected in 1 ml fractions rather than 2 ml fractions. The protocol was otherwise the same. The results are shown in FIGS. 5A, 5B and 6.

Results Observation of cell progression after infection: Day 2: 100% confluent; Day 4: 90% confluent—10% detachment; Day 7: 60% lysis; Day 8: 60-70% lysis; Day 9: Same on day 8; collected on day 10.

Conclusion A9 cells produced 9.75 log TCID ₅₀ / ml in untreated cell lysate. The high product titer resulted in a final virus stock concentration of 10.5 log TCID ₅₀ / ml. SDS-PAGE showed that the viral product from A9 growth had more protein than 324K lysate. However, despite the appearance of more protein in the final virus stock (which may be a viral protein), the final purity as measured by protein / infectious virus was about 2-5 fg protein / TCID _50. It was. Given that the medium may still be serum-free and the cellular protein background may be assumed to be equivalent, this may be a viral protein (and thus a higher viral titer result).

Example 3: Evaluation of hydraulic performance of various standard CTO spike stock solutions (see FIGS. 8-9)
Three CTO MVM spike stocks prepared by different methods were evaluated for their hydrodynamic performance when spiked into a monoclonal antibody (Mab) feed and processed through Viresolve Pro. The crude preparation was only clarified to remove cell debris and subjected to further purification. The ultracentrifugation preparation was purified by pelleting the virus in the infected cell culture lysate by ultracentrifugation, discarding the supernatant, and then resuspending the virus in PBS. The clarified infected cell culture lysate was passed through a membrane adsorber with positive charge Q chemistry to produce a membrane bound / elution purified preparation. The virus bound to the membrane, which was then eluted with a phosphate buffer containing 400 mM NaCl. MVM preparations made by ultracentrifugation and membrane-bound / elution purification methods are sold by CTO to customers for use in spike tests.

The target spike level for all three stocks was 1.4 × 10 ⁵ TCID ₅₀ / ml. Each condition was executed in duplicate (Run # 1 and Run # 2). The actual titer achieved with the spiked feed was: crude product: 2 × 10 ⁵ TCID ₅₀ / ml; after ultracentrifugation: 4 × 10 ⁴ TCID ₅₀ / ml; and after membrane binding / elution purification: 6 × 10 ⁴ TCID ₅₀ / ml. In particular, the hydrodynamic performance of the membrane-bound / eluted purified preparation was equivalent to that of the crude crude virus. In this comparison, the ultracentrifugation preparation performed best among the three CTO preparations, and the throughput was similar to the non-spike feed (at a spike level of 4 × 10 ⁴ TCID ₅₀ / ml). However, as explained in the above example, the virus preparation of the present invention performed significantly better than the CTO virus preparation (see, eg, FIG. 7).

  Protein was quantified by Micro BCA assay. * N. d. Not examined. Media components present in the crude virus stock interfere with the Micro BCA protein assay. DNA was quantified by PicoGreen® assay. As can be seen from Table 5, the CTO virus preparation has a significantly higher protein and DNA content.

It has been found that the amount of protein / virus directly correlates with the possibility that the virus preparation causes filter fouling. In addition to the assays performed above, three standard CTO MVM preparations and MVM preparations according to the method of the invention were analyzed by SDS-PAGE gel and silver staining analysis. FIG. 9 is a photograph of a silver stained 4-15% gradient SDS-PAGE gel. Each sample lane was loaded with 10 μl of sample. Lane 1: molecular weight marker; Lane 2: CTO crude MVM preparation (2.3 × 10 ⁷ TCID ₅₀ / ml); Lane 3: CTO ultracentrifugated purified MVM preparation (1.8 × 10 ⁷ TCID ₅₀ / ml); Lane 4: CTO Q membrane binding / elution purified MVM preparation (2.3 × 10 ⁷ TCID ₅₀ / ml); Lane 5: MVM preparation using the method of the present invention (6.5 × 10 ⁸ TCID ₅₀ / ml). ); Lane 6: molecular weight marker.

  As shown in FIG. 9, each of the CTO virus preparations contains the total number of proteins present (see protein band numbers and protein smears in each lane), and the total amount of protein per sample (as total staining amount). Proved) was significantly higher. In contrast, the methods of the present invention resulted in virus preparations with minimal protein content, both quantitatively and qualitatively, as described herein.

Example 4: Comparison of various cation exchange media used for flow-through MVM virus stock purification MVM virus stock (previously purified by buffer exchange to TNE and ultracentrifugation) in various cation exchange media I let it pass. The flow-through material was then assayed for virus titer and protein content by MicroBCA assay according to the manufacturer's protocol. All devices listed in Table 6 removed large amounts of protein impurities while maintaining the titer of the virus preparation. Millipore CEX (a membrane adsorber comprising AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) crosslinked with MBAm (N, N′-methylenebisacrylamide) on a polyethersulfone (PES) matrix) and Sartobind S15 (Sartorius) is both a cation exchange membrane adsorber. Pro Res-S (Millipore) is a monodisperse polymethacrylate strong cation exchange resin.

  As shown in Table 6, a variety of cation exchange media can be used in a flow-through manner to make high purity virus solutions using the methods as described herein.

Example 5: MVM Stock in Various Buffers Treated Using Cation Exchange (CEX) Membrane Adsorber MVM stock in various buffers was loaded onto a polyethersulfone (PES) matrix with MBAm (N, The cation exchange (CEX) membrane adsorbent containing AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) cross-linked by N′-methylenebisacrylamide) was used for the flow-through treatment. As shown in Table 7, removal of protein impurities was achieved in each of these conditions without significant loss of virus titer.

  The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

  Although the invention has been illustrated and described in detail with reference to exemplary embodiments thereof, those skilled in the art will recognize that the implementation can be made without departing from the scope of the invention as encompassed by the appended claims. It will be understood that various changes in form and detail may be made in form.

Claims (1)

A method for evaluating a mouse minut virus (MMV) clearance process comprising:
(A) a sample step spiked with MMV solution, making it a test sample, wherein said solution comprises a protein concentration of 2~ 17fg / TCID _{5 0,} the MMV solution comprises a flow-through chromatography Prepared by the method,
(B) testing the scaled MMV clearance process using the test sample; and (c) analyzing the results of the MMV clearance process;
A method wherein the amount of MMV clearance determines the MMV removal capability of the MMV clearance process, thereby evaluating the MMV clearance process.

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